The biomaterials formed by polymers play a central role in regenerative medicine since they provide temporary three-dimensional anchors for the adhesion, proliferation and the differentiation of transplanted cells. This three-dimensional nature provides a suitable platform for intercellular communication and the relationship of the cells with the components of the biomaterial. The biointeraction occurring between the matrix and the cells over time determines the proliferative capacity of the cells, their organization for the formation of a new tissue, their differentiation and the secretion of signaling molecules which direct the regenerative process (Dawson et al., 2008).
In order for these phenomena to occur, it is necessary for the biomaterial to remain in the site of application for a limited time until its reabsorption, conserving its structure long enough for a suitable cellular action with regenerative consequences.
A specific type of biomaterial, hydrogels, has a number of properties that make them suitable for their application in tissue engineering.
Hydrogels are structures formed by interconnected hydrophilic polymers of a natural or synthetic nature, with the capacity to contain a large amount of water inside their structure, from 10-20% up to hundreds of times their own weight. These gels show a semi-solid morphology the three-dimensional lattice of which is presented as an ideal candidate for forming a structural matrix capable of acting as a support. This three-dimensional structure can be formed by both physical crosslinking and by chemical crosslinking. Physical crosslinking leads to reversible hydrogels the structure of which can be reversed according to the end application, whereas chemical crosslinking leads to permanent hydrogels the structure of which will be maintained through the entire application (Coburn et al., 2007). Therefore, hydrogels are polymer materials (of a natural or synthetic nature) crosslinked in the form of a three-dimensional network which swell in contact with water, forming soft elastic materials, and which retain a significant fraction thereof in their structure without dissolving.
Hydrogels have a series of particular characteristics, such as:                1. Hydrophilic nature: due to the presence in their structure of water-soluble groups (—OH, —COOH, —CONH2, —CONH, SO3H). They have a high water content similar to that of live tissues (Elisseeff et al., 2005).        2. Insoluble in water: due to the existence of a three-dimensional polymer network in their structure.        3. They have a smooth and elastic consistency which is determined by the hydrophilic starting monomer and the low crosslinking density of the polymer.        
They have the capacity to swell in the presence of water or aqueous solutions, considerably increasing their volume until reaching a chemical-physical equilibrium, but without losing their form. This capacity to swell provides an aqueous microenvironment comparable to that which the cells are subjected in soft tissues. The presence of water and of a porous structure also allows the flow of low molecular weight solutes and of nutrients that are crucial and essential for cell viability, as well as the transport of cell wastes outside the hydrogel (Torres et al., 2000).
The umbilical cord is a highly vascularized structure with an important cell component. The cells and the vascular system are integrated in a gelatinous connective tissue called Wharton's jelly (WJ). WJ contains a low amount of cells and high levels of extracellular matrix, primarily made up of collagen, hyaluronic acid and sulfated glycosaminoglycans.
Glycosaminoglycans (GAGs), also referred to as mucopolysaccharides, are heteropolysaccharides found in organisms bound to a protein nucleus forming macromolecules referred to as proteoglycans. These can be found on the surfaces of cells or in the extracellular matrix and carry out important functions for cell-cell and cell-extracellular matrix interactions. They are in sulfated and non-sulfated form and the common characteristic of these molecules is their composition in a repeated sequence of disaccharides formed by two different sugars: one of them is usually a hexuronate while the other one is a hexosamine. The configurational variation in the bonding of the disaccharides and the position of sulfation leads to an increase of the diversity in the physical and chemical properties of these chains. The high sulfate content and the presence of uronic acid confers to GAGs a large negative charge, so the large amount of GAGs in WJ make this tissue be extremely hydrated.
There are several types of GAGs, which are directly involved in basic cell functions, not only due to their structure, but also because they are anchor sites for several cell signaling molecules.
Hyaluronic acid is the most abundant GAG in WJ. It is the only non-sulfated member of the GAG family which functions in vivo like a free carbohydrate, its structure consisting of repeats of a disaccharide: D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine (Goa et al., 1994; Laurent et al., 1992). It is synthesized by several cell types and is secreted into the extracellular space where it interacts with other components of the extracellular matrix to create the support and protection structure surrounding the cells (Collins et al., 2008). It is a large, polyanionic linear polymer, and a single molecule can have a molecular weight of 100,000 to 5.106 Da (Toole et al., 2004; Bertolami et al., 1992). It has a coiled structure taking up a large volume, leading to high viscosity solutions. The individual molecules of hyaluronic acid associate with one another, forming networks or lattices. In developing tissues, hyaluronic acid is considered the main structural macromolecule involved in cell proliferation and migration.
Hyaluronic acid has been involved in several processes, such as vascularization, morphogenesis, general integrity and repair of the extracellular matrix. It is known that a large amount of hyaluronic acid contained in amniotic fluid favors the repair of fetal wounds (Longaker et al., 1989). Variations in its molecular properties between healthy skin and scars have furthermore been observed, hyaluronic acid of normal scars certainly being different from that of hypertrophic scars (Ueno et al., 1992).
Chondroitin sulfate is a linear polymer formed by a D-glucuronic acid dimer and N-acetylgalactosamine repeat. Its usefulness has been tested in therapies targeted against joint diseases by means of inhibiting the activity of the enzymes responsible for the degradation of the matrix of the cartilage components. It would also act as an anti-inflammatory by means of the inhibition of the complement and is useful in the treatment of thromboembolic disorders, in surgery and opthalmological clinics.
Dermatan sulfate, also known as chondroitin sulfate B, is a potent anti-coagulant due to its selective inhibitory effect on thrombin through heparin cofactor II, being very effective in vivo due to its lower hemorrhagic risk (Trowbridge et al., 2002).
Glycosaminoglycans in general, and heparin in particular, have the capacity to modulate plasma cascade activity, enhancing the inhibition of the intrinsic coagulation pathway and inhibiting the classic complement activation pathway at different points (Rabenstein, 2001). Other known functions of the heparin are the inhibition of angiogenesis, humoral growth and its antiviral activity.
Heparan sulfate has a structure that is closely related to heparin. It is widely distributed in animal tissues and among its functions, cell adhesion and the regulation of cell proliferation stand out. It has a protective effect against the degradation of proteins, regulating their transport through the basement membrane and also intervening in the internalization thereof (Rabenstein, 2001).
There are several patent documents relating to mucopolysaccharides obtained from human or animal origin. Document U.S. Pat. No. 3,887,703 relates to mixtures of mucopolysaccharides obtained from the cutaneous teguments and umbilical cords of the fetus of a cow or sheep. The only example that uses an umbilical cord is of a cow fetus 1-9 months old and it does not mention that the membrane or the vessels are eliminated since the first operation is grinding under 10° C. The individual mucopolysaccharides forming the mixtures or the amounts present are not mentioned; the active products are identified by the amount of hexosamines that are present in the mixture. Compositions in both injectable and oral ingestion forms for the treatment of oily scalp and hair and for inflammations are prepared with the extracts.
Patent document U.S. Pat. No. 5,814,621 relates to a composition essentially consisting of a drug which is more soluble in an organic solvent-water mixture than in water, and a mucopolysaccharide forming part of a drug, in which crystals or particles of the drug are distributed on the surface of the particles of the mucopolysaccharide and in which said drug dissolves in water more quickly than if it were alone. Said composition can be in the form of granules.
Patent application WO 2008/021391 A1 describes biomaterials comprising the umbilical cord membrane. Furthermore, it can additionally comprise one or more umbilical cord vessels and/or Wharton's jelly. The biomaterial is preferably dry and can be flat, tubular or shaped to fit a particular structure. The invention also provides methods of making the biomaterial comprising at least one layer of the umbilical cord membrane, as well as the methods for obtaining said biomaterials and the use thereof for repairing tissues or organs.
The description characterizes the biomaterial from the umbilical cord. It describes that the composition of said material comprises collagen (type I, III and IV, these being 75-80% of the percentage of the matrix of the biomaterial), fibronectin and glycosaminoglycans.
It is also mentioned that the biomaterial can also comprise collagen that does not come from umbilical cords and has a commercial origin, or it has been isolated from other tissues and methods known in the state of the art. The authors also add that the biomaterial can comprise non-structural compounds such as growth factors, hormones, antibiotics, immunomodulatory factors, etc.
Spanish patent ES 8600613 describes a process for the treatment of body tissues, for separating cell membranes, nucleic acids, lipids and cytoplasmic components and forming an extracellular matrix the main component of which is collagens, and for making the body tissue suitable for being used as a body graft, comprising extracting said tissue with at least one detergent while at the same time it is maintained with a size and shape suitable for the grafting thereof in the body.
Patent document ES 2 180 653 T3 describes methods for transforming biological materials into substances which have experienced autolysis for eliminating at least 70% of the cells and methods for the treatment of said material for inhibiting its mineralization after implantation in a human or animal. It claims that the starting biological material can be, among others, the umbilical cord; although it specifically relates to an aortic valve of a pig. Nevertheless, the description does not contain any detail with respect to carrying it out with umbilical cord. The resulting biomaterial is used to create a bioprosthetic heart valve.
Patent document U.S. Pat. No. 4,240,794 relates to preparing human or other animal umbilical cords for their use as a vascular replacement. The document specifically describes a technique for dehydrating the umbilical cord in alcohol followed by a method for fixing it in the desired configuration. It is described that once the umbilical cord has been cleaned of possible remains of other tissues, it is mounted on a mandrel and immersed in a specific ethyl alcohol solution for the time necessary for it to dehydrate. After dehydration, the cord is immersed in a 1% aldehyde solution for fixing.
Patent document FR 2,563,727 describes a method for producing a skin graft from deprogrammed connective tissue impregnated with Wharton's jelly and stored at freezing temperatures. The authors describe a device which is anchored to the umbilical tissue and it is expanded by means of a cannula which injects compressed air. It is described that the umbilical cord is then cut and isolated but the product resulting from this process is not made up of WJ exclusively.
There are patent documents which use umbilical cord to obtain cells of interest, for which purpose they carry out processes for separating Wharton's jelly and eliminating it, thus obtaining said cells. For example, PCT document 98/17791 describes the isolation of pre-chondrocytes from the umbilical cord, which are subsequently used therapeutically to produce cartilage. Similarly, in document WO 2004/072273 A1 progenitor cells are extracted from Wharton's jelly that lies within the perivascular region of the umbilical cord and are used to repair human tissues.
However, there is no document that mentions a biomaterial formed by GAGs located in Wharton's jelly of the human umbilical cord, free of human umbilical cord membrane and blood vessels, which can form a hydrogel that adapts to the necessary viscosity characteristics, etc., to be used in different human pathologies.
Therefore, the biomaterial of the present invention is made up exclusively of the GAGs forming the extracellular matrix of the umbilical cord referred to as WJ. The extracellular matrix is a complex and specific biological substance of tissue. The extracellular matrix derived from blood vessels of the urinary bladder is completely different from that derived from the dermis (Hiles & Hodde, 2006). Thus, although several attempts to synthesize extracellular matrix are known in the literature, an exact composition that simulates the natural conditions of a certain tissue has not been achieved.
The biomaterial developed in the present invention offers a three-dimensional structure which allows the use thereof as a base matrix for tissue engineering and furthermore, when applied directly or with cells, in a pathology, it intervenes in the regenerative process, exerting a call effect on the cells of the tissue itself and providing a favorable environment for the activation of cell processes.
WJ is characterized in that it contains a very low number of cells and, nevertheless, a large amount of extracellular matrix (collagen and GAGs). In other words, the cells found in WJ are highly stimulated and are capable of producing high levels of matrix. This is due to the fact that large amounts of growth factors accumulate in WJ, including transforming growth factor beta (TGF-β), insulin-like growth factor type 1 (IGF-I), fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). These growth factors carry out their cell activity regulatory role by means of bonding to specific receptors, some of which are in the various GAGs making up WJ. These growth factors control cell proliferation, differentiation and the synthesis and remodeling of the extracellular matrix forming WJ. The large amount of synthesized matrix provides high mechanical resistance, elasticity and a high hydration capacity which is used to prevent the occlusion of the blood vessels caused by uterine contraction or fetal movements (Sobolewski et al., 2005).
Unlike other biomaterials, the biomaterial of the present invention is made up of a combination of different GAGs from the WJ of the umbilical cord. It is mostly made up of hyaluronic acid, but furthermore, unlike other GAG compounds, it contains dermatan sulfate, heparan sulfate, heparin, keratan sulfate, chondroitin-4-sulfate and chondroitin-6-sulfate. This combination of GAGs improves the bioactivity of the biomaterial, since each of them carries out cell behavior regulatory functions. For example, it is known that heparan sulfate and heparin are the main binding sites for FGF and EGF (Kanematsu et al., 2003; Ishihara et al., 2002), which protect them from proteolysis and allow local concentrations of these factors in the cell environment, creating the molecular microenvironment suitable for large cell activation (Malkowski et al., 2007).
The combination of GAGs present in this biomaterial provides a number of specific signaling molecule binding sites which will allow in the application site high activation of the cells of the tissue itself for the synthesis of high levels of extracellular matrix which will regenerate and repair the treated defect.
Furthermore, the origin of the biomaterial of the invention provides a natural structure of human origin of a non-immunogenic area, the elimination of which is integrated in the normal physiological cycles, which prevents the reactions of the biomaterials of animal origin or the side effects that some synthetic biomaterials may cause, such as inflammation, induration (hardening of organ tissues), onset of granulomas, necrosis in mucosae and tissue complications due to the toxic the substances used in the production thereof.
One of the most important functions of the GAGs in the umbilical cord is to provide strength, elasticity and resistance for protecting the vascular system located therein from external aggressions. In fact, the deficiency in the synthesis of these molecules is involved in important pathologies during pregnancy (Gogiel et al., 2005). Obtaining a biomaterial made up of the 7 different types of GAGs forming part of the umbilical cord would be capable of forming crosslinks between their fibers, simulating what occurs in the organism, and thus providing the strength, elasticity, resistance and compression similar to that conferred in the cord.